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BtuF (ABC Periplasmic Binding Protein)
The btuf gene encodes the vitamin B12 periplasmic binding protein of the BtuCD-F ABC transporter. The gene was cloned and expressed for functional analysis in a recent study of B12 metabolism in the Thermotogales ''species (1 ). This page focuses on the cloning and expression of the gene. Background ABC (ATP binding cassette) transporters are transport proteins located in the membrane of the cell. They are composed of 3 parts, an ATP-hydrolizing nucleotide-binding domain, a transmembrane domain and a binding protein (in the case of BtuCD-F, a periplasmic binding protein). ABC transporters are found across all organisms, from eukaryotes all the way to bacteria and archaea. The function of these proteins is almost as diverse. These transporters can movie nutrients into and out of cells, are involved in antibiotic resistance, and can help transport peptides across the membrane (2 ). The periplasmic binding protein of the BtuCD-F ABC transporter (BtuF) of certain Thermotogales'' species was produced to determine the ability of the protein to bind vitamin B12 and cobinamide (a precursor of vitamin B12), since the process of vitamin B12 salvaging and synthesis is still under investigation in these organisms. Production of the protein involved cloning of the btuf gene and expressing that gene in Escherichia coli cells through the use of a vector (1 ). pBAD TOPO Vector The pBAD TOPO vector from Invitrogen was used to express the btuf gene in competent E. coli ''cells. The pBAD TOPO vector has several regions of importance. pBR322 ori is the origin of replication for the vector. The ''araC ''gene encodes for the protein AraC, which allows for the transcription of the vector only when the sugar arabinose is present. The vector contains an ampicillin resistance gene, coding for beta-lactamase, which allows for selection of only the cells that contain the vector when grown on media containing ampicillin. The gene being expressed is inserted into a region of the vector, called a multiple cloning site, which contains 2 sequences downstream of the gene. The first sequence is the V5 epitope, which can be used to identify the protein after expression and translation with an antibody. The second sequence is a six histidine tag. This sequence encodes six histidine residues, which allows for the protein to be purified on a column of nickel-chelating resin, due to the high affininty of histidine for nickel. There is also an enterokinase (EK) cleavage site, which is a cleavage site just upstream of the gene of interest which is needed to remove the leader peptide after translation. The gene of interest is under the control of the ''ara''BAD promoter (shown in the diagram as pBAD), which AraC recognizes in the presence of arabinose. Finally, the TOPO portion of the vector name comes from the two topoisomerases linked to the site where the gene of interest is inserted. The topoisomerases allow for the gene to be inserted without the use of a DNA ligase (3 ). Expression and Purification Competent ''E. coli cells, meaning they are able to be transformed, are typically used with the pBAD TOPO vector. For expression of the BtuF protein, these cells were transformed with the cloned vector, specifically the One Shot TOP10 Chemically Competent E. coli cells from Invitrogen. This transformation was performed with the heat-shock technique (exposing the E. coli ''cells to a high temperature for a short period of time in the presence of the vector), but electroporation can also be used. Once transformed, the cells containing the vector can be selected for by growth on media containing ampicillin. After identification of the transformed cells, the production of the protein is induced by growing the transformed cells in the presence of arabinose. To purify the BtuF protein, a nickel sepharose column was ultimately used since the protein contained a histidine tag from the vector. Since this particular BtuF protein was from the hyperthermophilic ''Thermotogales species, the E. coli lysates were heated at a high temperature to denature any non-thermophilic proteins produced in the cells (1 ). This is a nice trick that works when purifying thermally stable proteins expressed in a non-thermophilic organism, however purification on a nickel sepharose column only would work for purifying non-thermophilic proteins expressed in the pBAD TOPO vector. Once purified, this BtuF protein was used in binding assays with vitamin B12 and precursor cobinamides (1 ). References 1. Butzin, N, et al. Thermotoga lettingae can salvage cobinamide to synthesize vitamin B12. Applied and Environmental Microbiology. (2013) 2. Jones, PM and George, AM. The ABC transporter structure and mechanism: perspectives on recent research. Cell. Mol. Life Sci. (2004) 3. pBAD TOPO TA Expression Kit User Manual. Invitrogen.